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Overexpression of the agouti-signaling protein (asip1), an endogenous melanocortin antagonist, under the control of a constitutive promoter in zebrafish [Tg(Xla.Eef1a1:Cau.Asip1]iim4] (asip1-Tg) increases food intake by reducing sensitivity of the central satiety systems and abolish circadian activity rhythms. The phenotype also shows increased linear growth and body weight, yet no enhanced aggressiveness in dyadic fights is observed. In fact, asip1-Tg animals choose to flee to safer areas rather than face a potential threat, thus suggesting a potential anxiety-like behavior (ALB). Standard behavioral tests, i.e., the open field test (OFT), the novel object test (NOT), and the novel tank dive test (NTDT), were used to investigate thigmotaxis and ALB in male and female zebrafish. Results showed that the asip1-Tg strain exhibited severe ALB in every test, mainly characterized by pronounced freezing behavior and increased linear and angular swimming velocities. asip1-Tg animals exhibited low central serotonin (5-HT) and dopamine (DA) levels and high turnover rates, thus suggesting that central monoaminergic pathways might mediate melanocortin antagonist-induced ALB. Accordingly, the treatment of asip1-Tg animals with fluoxetine, a selective serotonin reuptake inhibitor (SSRI), reversed the ALB phenotype in NTDT as well as 5-HT turnover. Genomic and anatomical data further supported neuronal interaction between melanocortinergic and serotonergic systems. These results suggest that inhibition of the melanocortin system by ubiquitous overexpression of endogenous antagonist has an anxiogenic effect mediated by serotonergic transmission.
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Ansiedade , Serotonina , Peixe-Zebra , Animais , Ansiedade/metabolismo , Ansiedade/psicologia , Masculino , Feminino , Serotonina/metabolismo , Animais Geneticamente Modificados , Comportamento Animal/efeitos dos fármacos , Comportamento Animal/fisiologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Fluoxetina/farmacologia , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Dopamina/metabolismo , Proteína Relacionada com Agouti/metabolismo , Proteína Relacionada com Agouti/genéticaRESUMO
Transposable elements (TEs) are mobile genetic modules of viral derivation that have been co-opted to become modulators of mammalian gene expression. TEs are a major source of endogenous dsRNAs, signaling molecules able to coordinate inflammatory responses in various physiological processes. Here, we provide evidence for a positive involvement of TEs in inflammation-driven bone repair and mineralization. In newly fractured mice bone, we observed an early transient upregulation of repeats occurring concurrently with the initiation of the inflammatory stage. In human bone biopsies, analysis revealed a significant correlation between repeats expression, mechanical stress and bone mineral density. We investigated a potential link between LINE-1 (L1) expression and bone mineralization by delivering a synthetic L1 RNA to osteoporotic patient-derived mesenchymal stem cells and observed a dsRNA-triggered protein kinase (PKR)-mediated stress response that led to strongly increased mineralization. This response was associated with a strong and transient inflammation, accompanied by a global translation attenuation induced by eIF2α phosphorylation. We demonstrated that L1 transfection reshaped the secretory profile of osteoblasts, triggering a paracrine activity that stimulated the mineralization of recipient cells.
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For almost two decades, clinicians have overlooked the diagnostic potential of CD34neg hematopoietic stem cells because of their limited homing capacity relative to CD34posHSCs when injected intravenously. This has contributed to the lack of appeal of using umbilical cord blood in HSC transplantation because its stem cell count is lower than bone marrow. The present study reveals that the homing and engraftment of CD34negHSCs can be improved by adding the Sialyl Lewis X molecule via α1,3-fucosylation. This unlocks the potential for using this more primitive stem cell to treat blood disorders because our findings show CD34negHSCs have the capacity to regenerate cells in the bone marrow of mice for several months. Furthermore, our RNA sequencing analysis revealed that CD34negHSCs have unique adhesion pathways, downregulated in CD34posHSCs, that facilitate interaction with the bone marrow niche. Our findings suggest that CD34neg cells will best thrive when the HSC resides in its microenvironment.
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Patients with Crohn's disease (CD) who smoke are known to have a worse prognosis than never-smokers and a higher risk for post-surgical recurrence, whereas patients who quit smoking after surgery have significantly lower post-operative recurrence. The hypothesis was that smoking induces epigenetic changes that impair the capacity of adipose stem cells (ASCs) to suppress the immune system. It was also questioned whether this impairment remains in ex-smokers with CD. ASCs were isolated from non-smokers, smokers and ex-smokers with CD and their interactions with immune cells were studied. The ASCs from both smokers and ex-smokers promoted macrophage polarization to an M1 pro-inflammatory phenotype, were not able to inhibit T- and B-cell proliferation in vitro and enhanced the gene and protein expression of inflammatory markers including interleukin-1b. Genome-wide epigenetic analysis using two different bioinformatic approaches revealed significant changes in the methylation patterns of genes that are critical for wound healing, immune and metabolic response and p53-mediated DNA damage response in ASCs from smokers and ex-smokers with CD. In conclusion, cigarette smoking induces a pro-inflammatory epigenetic signature in ASCs that likely compromises their therapeutic potential.
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Doença de Crohn , Humanos , Doença de Crohn/genética , Doença de Crohn/terapia , Fenótipo , Epigênese Genética , Células-Tronco/metabolismo , Fumar/efeitos adversosRESUMO
Anti-TNF biologics have been shown to markedly improve the quality of life for patients with Crohn's disease (CD), yet one-third of patients fail to benefit from this treatment. Patients with CD develop a characteristic wrapping of visceral adipose tissue (VAT) in the inflamed intestinal area, termed creeping fat, and it is known that adipose tissue expansion influences the efficacy of anti-TNF drugs. We questioned whether anti-TNF therapies impact the creeping fat in CD, which might affect the outcome of the disease. Adipose tissue biopsies were obtained from a cohort of 14 patients with CD that received anti-TNF drugs and from 29 non-anti-TNF-treated patients (control group) matched by sex, age, and body mass index undergoing surgical interventions for symptomatic complications. We found that anti-TNF therapies restored adipose tissue morphology and suppressed immune cell infiltration in the creeping fat. Additionally, anti-TNF treatments appeared to markedly improve the pro-inflammatory phenotype of adipose-tissue macrophages and adipose-tissue-derived stem cells. Our study provides evidence that anti-TNF medications influence immune cells and progenitor cells in the creeping of patients with CD, suppressing inflammation. We propose that perilesional VAT should be considered when administering anti-TNF therapy in patients with CD.
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Produtos Biológicos , Doença de Crohn , Tecido Adiposo/patologia , Produtos Biológicos/uso terapêutico , Humanos , Inflamação/tratamento farmacológico , Infliximab/uso terapêutico , Qualidade de VidaRESUMO
Constitutive heterochromatin is responsible for genome repression of DNA enriched in repetitive sequences, telomeres, and centromeres. During physiological and pathological premature aging, heterochromatin homeostasis is profoundly compromised. Here, we showed that LINE-1 (Long Interspersed Nuclear Element-1; L1) RNA accumulation was an early event in both typical and atypical human progeroid syndromes. L1 RNA negatively regulated the enzymatic activity of the histone-lysine N-methyltransferase SUV39H1 (suppression of variegation 3-9 homolog 1), resulting in heterochromatin loss and onset of senescent phenotypes in vitro. Depletion of L1 RNA in dermal fibroblast cells from patients with different progeroid syndromes using specific antisense oligonucleotides (ASOs) restored heterochromatin histone 3 lysine 9 and histone 3 lysine 27 trimethylation marks, reversed DNA methylation age, and counteracted the expression of senescence-associated secretory phenotype genes such as p16, p21, activating transcription factor 3 (ATF3), matrix metallopeptidase 13 (MMP13), interleukin 1a (IL1a), BTG anti-proliferation factor 2 (BTG2), and growth arrest and DNA damage inducible beta (GADD45b). Moreover, systemic delivery of ASOs rescued the histophysiology of tissues and increased the life span of a Hutchinson-Gilford progeria syndrome mouse model. Transcriptional profiling of human and mouse samples after L1 RNA depletion demonstrated that pathways associated with nuclear chromatin organization, cell proliferation, and transcription regulation were enriched. Similarly, pathways associated with aging, inflammatory response, innate immune response, and DNA damage were down-regulated. Our results highlight the role of L1 RNA in heterochromatin homeostasis in progeroid syndromes and identify a possible therapeutic approach to treat premature aging and related syndromes.
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Senilidade Prematura , Síndrome de Cockayne , Proteínas Imediatamente Precoces , Progéria , Senilidade Prematura/genética , Animais , Antígenos de Diferenciação , Heterocromatina , Histonas/metabolismo , Humanos , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Elementos Nucleotídeos Longos e Dispersos , Lisina/metabolismo , Camundongos , Fenótipo , Progéria/genética , RNA , Telômero/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
The histone demethylase KDM1A is a multi-faceted regulator of vital developmental processes, including mesodermal and cardiac tube formation during gastrulation. However, it is unknown whether the fine-tuning of KDM1A splicing isoforms, already shown to regulate neuronal maturation, is crucial for the specification and maintenance of cell identity during cardiogenesis. Here, we discovered a temporal modulation of ubKDM1A and KDM1A+2a during human and mice fetal cardiac development and evaluated their impact on the regulation of cardiac differentiation. We revealed a severely impaired cardiac differentiation in KDM1A-/- hESCs that can be rescued by re-expressing ubKDM1A or catalytically impaired ubKDM1A-K661A, but not by KDM1A+2a or KDM1A+2a-K661A. Conversely, KDM1A+2a-/- hESCs give rise to functional cardiac cells, displaying increased beating amplitude and frequency and enhanced expression of critical cardiogenic markers. Our findings prove the existence of a divergent scaffolding role of KDM1A splice variants, independent of their enzymatic activity, during hESC differentiation into cardiac cells.
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The Plant Resistance Genes database (PRGdb; http://prgdb.org/prgdb4/) has been greatly expanded, keeping pace with the increasing amount of available knowledge and data (sequenced proteomes, cloned genes, public analysis data, etc.). The easy-to-use style of the database website has been maintained, while an updated prediction tool, more data and a new section have been added. This new section will contain plant resistance transcriptomic experiments, providing additional easy-to-access experimental information. DRAGO3, the tool for automatic annotation and prediction of plant resistance genes behind PRGdb, has been improved in both accuracy and sensitivity, leading to more reliable predictions. PRGdb offers 199 reference resistance genes and 586.652 putative resistance genes from 182 sequenced proteomes. Compared to the previous release, PRGdb 4.0 has increased the number of reference resistance genes from 153 to 199, the number of putative resistance genes from 177K from 76 proteomes to 586K from 182 sequenced proteomes. A new section has been created that collects plant-pathogen transcriptomic data for five species of agricultural interest. Thereby, with these improvements and data expansions, PRGdb 4.0 aims to serve as a reference to the plant scientific community and breeders worldwide, helping to further study plant resistance mechanisms that contribute to fighting pathogens.
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Bases de Dados Genéticas , Resistência à Doença/genética , Doenças das Plantas/genética , Plantas/genética , Genoma de Planta/genética , Anotação de Sequência Molecular , Doenças das Plantas/classificação , Plantas/classificação , Transcriptoma/genéticaRESUMO
Klinefelter syndrome (KS) is the most prevalent aneuploidy in males and is characterized by a 47,XXY karyotype. Less frequently, higher grade sex chromosome aneuploidies (HGAs) can also occur. Here, using a paradigmatic cohort of KS and HGA induced pluripotent stem cells (iPSCs) carrying 49,XXXXY, 48,XXXY, and 47,XXY karyotypes, we identified the genes within the pseudoautosomal region 1 (PAR1) as the most susceptible to dosage-dependent transcriptional dysregulation and therefore potentially responsible for the progressively worsening phenotype in higher grade X aneuploidies. By contrast, the biallelically expressed non-PAR escape genes displayed high interclonal and interpatient variability in iPSCs and differentiated derivatives, suggesting that these genes could be associated with variable KS traits. By interrogating KS and HGA iPSCs at the single-cell resolution we showed that PAR1 and non-PAR escape genes are not only resilient to the X-inactive specific transcript (XIST)-mediated inactivation but also that their transcriptional regulation is disjointed from the absolute XIST expression level. Finally, we explored the transcriptional effects of X chromosome overdosage on autosomes and identified the nuclear respiratory factor 1 (NRF1) as a key regulator of the zinc finger protein X-linked (ZFX). Our study provides the first evidence of an X-dosage-sensitive autosomal transcription factor regulating an X-linked gene in low- and high-grade X aneuploidies.
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Zebrafish have the ability to regenerate damaged cells and tissues by activating quiescent stem and progenitor cells or reprogramming differentiated cells into regeneration-competent precursors. Proliferation among the cells that will functionally restore injured tissues is a fundamental biological process underlying regeneration. Midkine-a is a cytokine growth factor, whose expression is strongly induced by injury in a variety of tissues across a range of vertebrate classes. Using a zebrafish Midkine-a loss of function mutant, we evaluated regeneration of caudal fin, extraocular muscle and retinal neurons to investigate the function of Midkine-a during epimorphic regeneration. In wildtype zebrafish, injury among these tissues induces robust proliferation and rapid regeneration. In Midkine-a mutants, the initial proliferation in each of these tissues is significantly diminished or absent. Regeneration of the caudal fin and extraocular muscle is delayed; regeneration of the retina is nearly completely absent. These data demonstrate that Midkine-a is universally required in the signaling pathways that convert tissue injury into the initial burst of cell proliferation. Further, these data highlight differences in the molecular mechanisms that regulate epimorphic regeneration in zebrafish.
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Midkina/metabolismo , Regeneração/fisiologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Nadadeiras de Animais/fisiologia , Animais , Animais Geneticamente Modificados/metabolismo , Diferenciação Celular , Proliferação de Células , Midkina/genética , Mutagênese , Neuroglia/citologia , Neuroglia/metabolismo , Músculos Oculomotores/fisiologia , Neurônios Retinianos/fisiologia , Proteínas de Peixe-Zebra/genéticaRESUMO
BACKGROUND: Crohn's disease (CD) is characterized by persistent inflammation and ulceration of the small or large bowel, and expansion of mesenteric adipose tissue, termed creeping fat (CF). We previously demonstrated that human adipose-derived stem cells (hASCs) from CF of patients with CD exhibit dysfunctional phenotypes, including a pro-inflammatory profile, high phagocytic capacity, and weak immunosuppressive properties. Importantly, these phenotypes persist in patients in remission and are found in all adipose depots explored including subcutaneous fat. We hypothesized that changes in hASCs are a consequence of epigenetic modifications. METHODS: We applied epigenome-wide profiling with a methylation array (Illumina EPIC/850k array) and gene expression analysis to explore the impact of CD on the methylation signature of hASCs isolated from the subcutaneous fat of patients with CD and healthy controls (n = 7 and 5, respectively; cohort I). Differentially methylated positions (p value cutoff < 1 × 10-4 and ten or more DMPs per gene) and regions (inclusion threshold 0.2, p value cutoff < 1 × 10-2 and more than 2 DMRs per gene) were identified using dmpfinder and Bumphunter (minfi), respectively. Changes in the expression of differentially methylated genes in hASCs were validated in a second cohort (n = 10/10 inactive and active CD and 10 controls; including patients from cohort I) and also in peripheral blood mononuclear cells (PBMCs) of patients with active/inactive CD and of healthy controls (cohort III; n = 30 independent subjects). RESULTS: We found a distinct DNA methylation landscape in hASCs from patients with CD, leading to changes in the expression of differentially methylated genes involved in immune response, metabolic, cell differentiation, and development processes. Notably, the expression of several of these genes in hASCs and PBMCs such as tumor necrosis factor alpha (TNFA) and PR domain zinc finger protein 16 (PRDM16) were not restored to normal (healthy) levels after disease remission. CONCLUSIONS: hASCs of patients with CD exhibit a unique DNA methylation and gene expression profile, but the expression of several genes are only partially restored in patients with inactive CD, both in hASCs and PBMCs. Understanding how CD shapes the functionality of hASCs is critical for investigating the complex pathophysiology of this disease, as well as for the success of cell-based therapies. Human adipose-stem cells isolated from subcutaneous fat of patients with Crohn's disease exhibit an altered DNA methylation pattern and gene expression profile compared with those isolated from healthy individuals, with immune system, cell differentiation, metabolic and development processes identified as the main pathways affected. Interestingly, the gene expression of several genes involved in these pathways is only partially restored to control levels in patients with inactive Crohn's disease, both in human adipose-stem cells and peripheral blood mononuclear cells. Understanding how Crohn's disease shapes the functionality of human adipose-stem cells is critical for investigating the complex pathophysiology of this disease, as well as for the success of cell-based therapies.
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Tecido Adiposo/química , Doença de Crohn/genética , Metilação de DNA , Epigenômica/métodos , Redes Reguladoras de Genes , Estudos de Casos e Controles , Técnicas de Cultura de Células , Epigênese Genética , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Células-Tronco/químicaRESUMO
Zebrafish extraocular muscles regenerate after severe injury. Injured myocytes dedifferentiate to a mesenchymal progenitor state and reenter the cell cycle to proliferate, migrate, and redifferentiate into functional muscles. A dedifferentiation process that begins with a multinucleated syncytial myofiber filled with sarcomeres and ends with proliferating mononucleated myoblasts must include significant remodeling of the protein machinery and organelle content of the cell. It turns out that autophagy plays a key role early in this process, to degrade the sarcomeres as well as the excess nuclei of the syncytial multinucleated myofibers. Because of the robustness of the zebrafish reprogramming process, and its relative synchrony, it can serve as a useful in vivo model for studying the biology of autophagy. In this chapter, we describe the surgical muscle injury model as well as the experimental protocols for assessing and manipulating autophagy activation.
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Autofagia , Músculos Oculomotores/lesões , Músculos Oculomotores/fisiologia , Regeneração , Animais , Ciclo Celular , Desdiferenciação Celular , Proliferação de Células , Reprogramação Celular , Imuno-Histoquímica , Microscopia Eletrônica de Transmissão , Modelos Biológicos , Músculos Oculomotores/metabolismo , Peixe-ZebraRESUMO
Insulin-like growth factors (Igfs) are key regulators of key biological processes such as embryonic development, growth, and tissue repair and regeneration. The role of Igf in myogenesis is well documented and, in zebrafish, promotes fin and heart regeneration. However, the mechanism of action of Igf in muscle repair and regeneration is not well understood. Using adult zebrafish extraocular muscle (EOM) regeneration as an experimental model, we show that Igf1 receptor blockage using either chemical inhibitors (BMS754807 and NVP-AEW541) or translation-blocking morpholino oligonucleotides (MOs) reduced EOM regeneration. Zebrafish EOMs regeneration depends on myocyte dedifferentiation, which is driven by early epigenetic reprogramming and requires autophagy activation and cell cycle reentry. Inhibition of Igf signaling had no effect on either autophagy activation or cell proliferation, indicating that Igf signaling was not involved in the early reprogramming steps of regeneration. Instead, blocking Igf signaling produced hypercellularity of regenerating EOMs and diminished myosin expression, resulting in lack of mature differentiated muscle fibers even many days after injury, indicating that Igf was involved in late re-differentiation steps. Although it is considered the main mediator of myogenic Igf actions, Akt activation decreased in regenerating EOMs, suggesting that alternative signaling pathways mediate Igf activity in muscle regeneration. In conclusion, Igf signaling is critical for re-differentiation of reprogrammed myoblasts during late steps of zebrafish EOM regeneration, suggesting a regulatory mechanism for determining regenerated muscle size and timing of differentiation, and a potential target for regenerative therapy.
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Músculos Oculomotores/fisiologia , Regeneração , Transdução de Sinais , Somatomedinas/metabolismo , Peixe-Zebra/fisiologia , Animais , Diferenciação Celular , Músculos Oculomotores/citologia , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
BACKGROUND: Tissue regeneration requires a series of steps, beginning with generation of the necessary cell mass, followed by cell migration into damaged area, and ending with differentiation and integration with surrounding tissues. Temporal regulation of these steps lies at the heart of the regenerative process, yet its basis is not well understood. The ability of zebrafish to dedifferentiate mature "post-mitotic" myocytes into proliferating myoblasts that in turn regenerate lost muscle tissue provides an opportunity to probe the molecular mechanisms of regeneration. RESULTS: Following subtotal excision of adult zebrafish lateral rectus muscle, dedifferentiating residual myocytes were collected at two time points prior to cell cycle reentry and compared to uninjured muscles using RNA-seq. Functional annotation (GAGE or K-means clustering followed by GO enrichment) revealed a coordinated response encompassing epigenetic regulation of transcription, RNA processing, and DNA replication and repair, along with protein degradation and translation that would rewire the cellular proteome and metabolome. Selected candidate genes were phenotypically validated in vivo by morpholino knockdown. Rapidly induced gene products, such as the Polycomb group factors Ezh2 and Suz12a, were necessary for both efficient dedifferentiation (i.e. cell reprogramming leading to cell cycle reentry) and complete anatomic regeneration. In contrast, the late activated gene fibronectin was important for efficient anatomic muscle regeneration but not for the early step of myocyte cell cycle reentry. CONCLUSIONS: Reprogramming of a "post-mitotic" myocyte into a dedifferentiated myoblast requires a complex coordinated effort that reshapes the cellular proteome and rewires metabolic pathways mediated by heritable yet nuanced epigenetic alterations and molecular switches, including transcription factors and non-coding RNAs. Our studies show that temporal regulation of gene expression is programmatically linked to distinct steps in the regeneration process, with immediate early expression driving dedifferentiation and reprogramming, and later expression facilitating anatomical regeneration.
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Desdiferenciação Celular/genética , Perfilação da Expressão Gênica , Células Musculares/citologia , Desenvolvimento Muscular/genética , Músculos/fisiologia , Regeneração/genética , Transcrição Gênica , Animais , Ontologia Genética , Fatores de Tempo , Peixe-ZebraRESUMO
Cell identity involves both selective gene activity and specialization of cytoplasmic architecture and protein machinery. Similarly, reprogramming differentiated cells requires both genetic program alterations and remodeling of the cellular architecture. While changes in genetic and epigenetic programs have been well documented in dedifferentiating cells, the pathways responsible for remodeling the cellular architecture and eliminating specialized protein complexes are not as well understood. Here, we utilize a zebrafish model of adult muscle regeneration to study cytoplasmic remodeling during cell dedifferentiation. We describe activation of autophagy early in the regenerative response to muscle injury, while blocking autophagy using chloroquine or Atg5 and Becn1 knockdown reduced the rate of regeneration with accumulation of sarcomeric and nuclear debris. We further identify Casp3/caspase 3 as a candidate mediator of cellular reprogramming and Fgf signaling as an important activator of autophagy in dedifferentiating myocytes. We conclude that autophagy plays a critical role in cell reprogramming by regulating cytoplasmic remodeling, facilitating the transition to a less differentiated cell identity.
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Autofagia , Reprogramação Celular , Citoplasma/metabolismo , Modelos Biológicos , Músculos Oculomotores/fisiologia , Regeneração , Peixe-Zebra/fisiologia , Animais , Apoptose/genética , Metabolismo Energético , Fatores de Crescimento de Fibroblastos/metabolismo , Regulação para Cima , Peixe-Zebra/genéticaRESUMO
Fibroblast growth factors (Fgfs) regulate critical biological processes such as embryonic development, tissue homeostasis, wound healing, and tissue regeneration. In zebrafish, Fgf signaling plays an important role in the regeneration of the spinal cord, liver, heart, fin, and photoreceptors, although its exact mechanism of action is not fully understood. Utilizing an adult zebrafish extraocular muscle (EOM) regeneration model, we demonstrate that blocking Fgf receptor function using either a chemical inhibitor (SU5402) or a dominant-negative transgenic construct (dnFGFR1a:EGFP) impairs muscle regeneration. Adult zebrafish EOMs regenerate through a myocyte dedifferentiation process, which involves a muscle-to-mesenchyme transition and cell cycle reentry by differentiated myocytes. Blocking Fgf signaling reduced cell proliferation and active caspase 3 levels in the regenerating muscle with no detectable levels of apoptosis, supporting the hypothesis that Fgf signaling is involved in the early steps of dedifferentiation. Fgf signaling in regenerating myocytes involves the MAPK/ERK pathway: inhibition of MEK activity with U0126 mimicked the phenotype of the Fgf receptor inhibition on both muscle regeneration and cell proliferation, and activated ERK (p-ERK) was detected in injured muscles by immunofluorescence and western blot. Interestingly, following injury, ERK2 expression is specifically induced and activated by phosphorylation, suggesting a key role in muscle regeneration. We conclude that the critical early steps of myocyte dedifferentiation in EOM regeneration are dependent on Fgf signaling.
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Envelhecimento/fisiologia , Desdiferenciação Celular , Fatores de Crescimento de Fibroblastos/metabolismo , Músculo Esquelético/fisiologia , Regeneração , Peixe-Zebra/fisiologia , Animais , Proliferação de Células , Proteínas de Fluorescência Verde/metabolismo , Sistema de Sinalização das MAP Quinases , Modelos Biológicos , Músculos Oculomotores , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismoRESUMO
PURPOSE: The purpose of this study was to characterize the injury response of extraocular muscles (EOMs) in adult zebrafish. METHODS: Adult zebrafish underwent lateral rectus (LR) muscle myectomy surgery to remove 50% of the muscle, followed by molecular and cellular characterization of the tissue response to the injury. RESULTS: Following myectomy, the LR muscle regenerated an anatomically correct and functional muscle within 7 to 10 days post injury (DPI). Following injury, the residual muscle stump was replaced by a mesenchymal cell population that lost cell polarity and expressed mesenchymal markers. Next, a robust proliferative burst repopulated the area of the regenerating muscle. Regenerating cells expressed myod, identifying them as myoblasts. However, both immunofluorescence and electron microscopy failed to identify classic Pax7-positive satellite cells in control or injured EOMs. Instead, some proliferating nuclei were noted to express mef2c at the very earliest point in the proliferative burst, suggesting myonuclear reprogramming and dedifferentiation. Bromodeoxyuridine (BrdU) labeling of regenerating cells followed by a second myectomy without repeat labeling resulted in a twice-regenerated muscle broadly populated by BrdU-labeled nuclei with minimal apparent dilution of the BrdU signal. A double-pulse experiment using BrdU and 5-ethynyl-2'-deoxyuridine (EdU) identified double-labeled nuclei, confirming the shared progenitor lineage. Rapid regeneration occurred despite a cell cycle length of 19.1 hours, whereas 72% of the regenerating muscle nuclei entered the cell cycle by 48 hours post injury (HPI). Dextran lineage tracing revealed that residual myocytes were responsible for muscle regeneration. CONCLUSIONS: EOM regeneration in adult zebrafish occurs by dedifferentiation of residual myocytes involving a muscle-to-mesenchyme transition. A mechanistic understanding of myocyte reprogramming may facilitate novel approaches to the development of molecular tools for targeted therapeutic regeneration in skeletal muscle disorders and beyond.
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Células Musculares/fisiologia , Músculos Oculomotores/fisiologia , Regeneração/fisiologia , Animais , Ciclo Celular , Seguimentos , Imuno-Histoquímica , Microscopia Eletrônica de Transmissão , Células Musculares/ultraestrutura , Mioblastos/fisiologia , Mioblastos/ultraestrutura , Músculos Oculomotores/cirurgia , Músculos Oculomotores/ultraestrutura , Peixe-ZebraRESUMO
Heat shock is a common technique for inducible gene expression system in a variety of organisms. Heat shock treatment of adult zebrafish is more involved and generally consists of manually transferring fish between housing rack tanks and preheated water tanks or the use of timed heaters in stand-alone aquaria. To avoid excessive fish handling and to take advantage of the continuous flow of a standard housing rack, proposed modifications consisted of installing an aquarium heater inside each tank, manually setting the heater to reach heat shocking temperatures (> 37°C) and, after that, testing that every tank responded equally. To address the limitations in the existing systems, we developed a novel modification of standard zebrafish housing racks to perform heat shock treatment in conditions of continuous water flow. By adding an extra manifold to the housing rack and connecting it to a recirculating bath to create a parallel water flow system, we can increase the temperature from standard conditions (28.5°C) to heat shock conditions with high precision (38.0-38.3°C, mean ± SD = 38.1°C ± 0.14°C) and minimal variation among experimental tanks (coefficient of variation [CV] = 0.04%). This means that there is virtually no need for laborious pretreatment calibrations or continuous adjustments to minimize intertank variation. To test the effectiveness of our design, we utilized this system to induce enhanced green fluorescent protein (EGFP) expression in hsp70-EGFP fish and performed a fin regeneration experiment with hsp70l:dnfgfr1-EGFP fish to confirm that heat-induced gene expression reached physiological levels. In summary, our newly described aquatic heat shock system minimizes effort during heat shock experiments, while ensuring the best water quality and fish welfare and facilitating large heat shock settings or the use of multiple transgenic lines for both research and teaching experiments.
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Bem-Estar do Animal , Aquicultura/métodos , Resposta ao Choque Térmico , Abrigo para Animais , Qualidade da Água , Peixe-Zebra/fisiologia , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/crescimento & desenvolvimento , Animais Geneticamente Modificados/fisiologia , Aquicultura/instrumentação , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
Juveniles of gilthead sea bream were fed with plant protein-based diets with fish oil (FO diet) or vegetable oils (66VO diet) as dietary lipid sources. No differences in growth performance were found between both groups, and fish with an average body mass of 65-70 g were crowded (90-100 kg/m(3)) to assess the stress response within the 72 h after the onset of stressor. The rise in plasma cortisol and glucose levels was higher in stressed fish of group 66VO (66VO-S) than in FO group (FO-S), but the former stressed group regained more quickly the cortisol resting values of the corresponding non-stressed diet group. The cell-tissue repair response represented by derlin-1, 75 kDa glucose-regulated protein and 170 kDa glucose-regulated protein was triggered at a lower level in 66VO-S than in FO-S fish. This occurred in concert with a long-lasting up-regulation of glucocorticoid receptors, antioxidant enzymes, enzyme subunits of the mitochondrial respiratory chain, and enzymes involved in tissue fatty acid uptake and ß-oxidation. This gene expression pattern allows a metabolic phenotype that is prone to "high power" mitochondria, which would support the replacement of fish oil with vegetable oils when theoretical requirements in essential fatty acids for normal growth are met by diet.
Assuntos
Aglomeração , Gorduras Insaturadas na Dieta/administração & dosagem , Hidrocortisona/metabolismo , RNA Mensageiro/biossíntese , Dourada/fisiologia , Estresse Fisiológico/fisiologia , Animais , Perfilação da Expressão Gênica , Homeostase/fisiologia , Hidrocortisona/sangue , Fígado/metabolismo , Fígado/fisiologia , RNA Mensageiro/análise , RNA Mensageiro/genética , Dourada/genética , Dourada/metabolismoRESUMO
The effects of fish oil (FO) substitution by 66% vegetable oils in a diet with already 75% vegetable protein (66VO) on adipose tissue lipid metabolism of gilthead sea bream were analysed after a 14-month feeding trial. In the last 3 months of the experiment, a FO diet was administrated to a 66VO group (group 66VO/FO) as a finishing diet. Hormone-sensitive lipase (HSL) activity was measured in adipose tissue and adipocyte size, and HSL, lipoprotein lipase and liver X receptor gene expression in isolated adipocytes, on which lipolysis and glucose uptake experiments were also performed. Lipolysis was measured after incubation with tumour necrosis factor-α (TNFα), linoleic acid, and two conjugated linoleic acid isomers. Glucose uptake was analysed after TNFα or insulin administration. Our results show that FO replacement increased lipolytic activity and adipocyte cell size. The higher proportion of large cells observed in the 66VO group could be involved in their observed lower response to fatty acid treatments and lower insulin sensitivity. The 66VO/FO group showed a moderate return to the FO conditions. Therefore, FO replacement can affect the morphology and metabolism of gilthead sea bream adipocytes which could potentially affect other organs such as the liver.